IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-18

Logistics Communications

Streptavidin Magnetic Beads, Take 3-4

• Trypsinized the beads, in 50uL solution that were retained from yesterday, for four hours @ 37[[:Category:{{{1}}}|{{{1}}}]].
• Ran gel of them, ladder, and p7308
  • Noticed when took the gel out for imaging that the buffer was below the gel slightly - didn't cover top
  • Imaging: NO BANDS AT ALL, not for the ladder or even p7308.
    • Suspect that the gel didn't run due to too-low-buffer.

• On the off-chance that the trypsin digest didn't work, used 2uL (of unknown activity concentration, see above) of proteinase K to try again to digest the streptavidin.
  • Incubated overnight at 37[[:Category:{{{1}}}|{{{1}}}]].

• Mixed c5.0.1 and other pre-working stocks and working stocks listed above.

• Folded 4 rxns each, at intermediate {Mgcl2}} of 20mM final concentration, of:
  • E(b)
  • F(b)
  • A lidless

12 Hours Later

• Ran gel of proteinase-K-ed final supernatant

• • Gel:

Lane	Component	Amount
1	1kb ladder	1uL (from stock of 1ug/uL)
2	p7308	9uL
3	c5.0.A (unbiotinylated barrel) wash-supernatant	38uL
4	c5.0.A (unbiotinylated barrel) final-supernatantc	38uL
5	5.0.8b wash-supernatant	38uL
6	c5.0.8b final-supernatant	38uL
7	c5.0."Eb" (inside biotinylation) wash-supernatant	38uL
8	c5.0."Eb" (inside biotinylation) final-supernatant	38uL
9	c5.0."Fb" (outside biotinylation) wash-supernatant	38uL
10	c5.0."Fb" (outside biotinylation) final-supernatant	38uL

• 

  2% agarose, 10mM MgCl2, 3uL EtBr, 1:10hr @ 80V

• • Results:
    • NOTHING in final-supernatant lanes again, indicating either:
      • (A) Second trypsinization (not able to be visualized because ran the gel without enough buffer) worked, leaving nothing on the streptavidin for the proteinase K to have digested.
      • (B) Proteinase K did not digest the streptavidin to elute.
    • Wash lanes were also dim - did not Speedvac for long enough.
      • Also, gel was older and had been left out of fridge overnight.
        • Plan: Try again with proteinase K as eluent, as there were so many problems with this trial (ie. "Eb" and "Fb", trypsinization first two times around)

Folding design 6

• Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5)
  • To mix pre-working stocks from plates, pipet 10 μL of each of the appropriate oligos into 1.5 mL tubes.
  • List of pre-working and working stock oligos: Table_of_c6.0_Working_Stocks